Method for producing pectin hydrolysis products

ABSTRACT

Methods for the production of pectin hydrolysis products, the pectin hydrolysis products produced in this manner, as well as their use are described.

This is division of application Ser. No. 12/416,803, filed Apr. 1, 2009,now U.S. Pat. No. 7,576,070, which was a division of application Ser.No. 10/416,347, filed Sep. 29, 2003.

FIELD OF THE INVENTION

The invention relates to a method for producing pectin hydrolysates, inparticular of a pharmaceutical or dietetic preparation for reducingand/or preventing the adhesion of pathogenic substances and organisms toeukaryotic cells, especially mammalian cells, or for inhibitinggalectin-3-mediated cell-cell and/or cell-matrix interactions leading tothe development of tumor diseases, methods for blocking the attachmentof pathogenic substances or organisms to eukaryotic cells, methods forinhibiting galectin-3-mediated cell-cell and/or cell-matrixinteractions, as well as pectin hydrolysates and preparations producedby using these methods.

BACKGROUND OF THE INVENTION

Pathogenic organisms, and also cell-damaging substances, first mustadhere to the surface of the target cell in order to be able to causeinfection or damage the attacked cell. This adhesion is mediated, forexample, by a ligand-receptor relationship, whereby glycostructures playan important role. If these glycostructures are blocked at the targetcell surface or at the ligand, an infection can be prevented.

Glycostructures also play an important role in the formation of tumorsand metastases (Liotta et al., Annu. Rev. Cell Biol., 55 (1986),1037-1057). The formation of tumors includes cellular interactionsmediated by cell surface components, in particular carbohydrate-bindingproteins. This mediates the adhesion of tumor cells by way of cellularadhesion molecules. Many stages in the formation of metastases alsoinclude cell-cell interactions or interactions between cells and theextracellular matrix (ECM) which are mediated by cell surfacecomponents. The extracellular matrix (ECM) consists mainly of laminin,fibronectin, and proteoglycanes, of which very many are glycosylated,and whose oligosaccharide side chains provide detection determinants forcellular adhesion molecules. Laminin is an N-bound glycoprotein withpoly-N-acetyl lactosamine sequences. Metastatic spread occurs whencirculating agglomerates of tumor cells, thrombocytes, and lymphocytesmake contact in capillaries by way of adhesion molecules with theendothelium. This contact provides the signal for opening theendothelial cell functions. As a result, the tumor cells are able tobind to receptors on the basal membrane by way of additional adhesionmolecules. After destroying the basal membrane, the tumor cells getdirect access to the stroma, whereby again interactions occur betweenthe laminin and fibronectin and the respective receptors, as was thecase in the primary tumor invasion.

Important representatives of the carbohydrate-binding proteins are thegalactoside-binding lectins galectin-1 and galectin-3 (Raz and Lotan,Cancer Metastasis Rev. 6 (1987), 433; Gabius, Biochim. Biophys. Acta,1071 (1991), 1). It is known that galectin-3 promotes the embolic tumordispersal in the circulation and increases the formation of metastases.Galectin-3 is expressed on the cell surface of many tumor cells, wherebythe galectin-3 expression increases with progressing tumor development.Galectin-3 is also expressed by activated macrophages and oncogenicallytransformed cells or metastasis cells. Galectin-3 has a high affinityfor oligosaccharides, which include polylactosamines, whereby galectin-3binds in particular to two glycoproteins that occur in the form ofseveral cell types, for example human colon cancer cells and humanbreast cancer cells. Another ligand for galectin-3 is, for example,laminin. Galectin-3, which is also expressed on the surface ofendothelial cells, is also involved in the adhesion of tumor cells toendothelial cells.

U.S. Pat. No. 5,834,442 describes methods for treating cancers inmammals, in particular for treating prostate cancers, where thetreatment of cancers, including the inhibition of the formation ofmetastases, is performed by oral administration of modified pectin,preferably water-soluble, pH-modified citrus pectin. To producepH-modified pectin, a pectin solution is depolymerized by increasing itspH value to 10.0 and then reducing the pH value to 3.0. The modifiedpectin has a molecular weight of approximately 1 to 15 kd. Rats thatwere administered modified citrus pectin in their drinking water showeda significantly reduced formation of lung metastases compared tountreated control groups. In vitro experiments demonstrated that theadhesion of galectin-3-expressing MLL endothelial cells to rat aorticendothelial cells (RAEC) was almost completely inhibited in the presenceof modified citrus pectin. Other experiments studied the effect ofpH-modified citrus pectin on the colonization of MLL endothelial cells.The ability of cells to grow in semi-solid medium (anchorageindependence) may be used as a criterion for cell transformation and theinvasive potential of cells, since cell growth in a semi-solid mediumrequires cell migration and colonization. It was hereby found thatmodified citrus pectin was able to significantly reduce both the numberof MLL colonies formed as well as their size. In the process, modifiedcitrus pectin appears to have more of a cytostatics effect than acytotoxic effect. The effect of modified citrus pectin on cell-cellinteractions and cell-matrix interactions based on carbohydrate-mediatedmechanisms, especially galectin-3-mediated interactions, were alsoinvestigated. It was found that, in contrast to non-modified citruspectin, modified citrus pectin inhibited the adhesion of B16-F1 melanomacells to laminin. Of laminin, it is known that it acts as a ligand forsoluble galectin-3.

From EP 0 716 605 B1 it is known that the adherence of pathogenicagents, such as, for example, E. coli, to cells, in particular toepithelial cells of the gastrointestinal and genitourinary tract can besubstantially (i.e., up to 90%) reduced with a specially prepared carrotsoup, bladder tea, coconut milk, etc. According to this document, thiseffect can be attributed to the pectins contained in these plantproducts, which are essentially chains of 1,4-α-glycosidically boundgalacturonides whose acid groups are esterified 20 to 80% with methanoland which, in addition to galacturonic acid, also may contain othersugar components, for example, glucose, galactose, xylose, andarabinose.

The document further describes that monogalacturonic acid shows noblocking of the adhesion, while a blocking of up top 91.7% or 84.6% canbe found with digalacturonide and trigalacturonide respectively. Thisdocument clearly determines that the monomer galacturonic acid does notblock the adhesion, and the desired blocking effect decreases along withan increasing molecular weight of the galacturonides. This means thatthe degree of polymerization of the desired galacturonides is DP 2 or 3.It is also required that the degree of esterification is <2%. The pectinhydrolysis products produced according to the method described in thisdocument contain only very small portions of the di- andtrigalacturonides designated as effective, however (approximately 12%related to the raw material). This production method wastes resourcesand causes environmental problems, because large quantities of unusablesecondary products are created and must be disposed of.

The technical problem underlying the present invention thereforeconsists of providing additional methods and means for fightinginfections and for reducing and/or preventing the adhesion of harmful,in particular pathogenic, substances and organisms to eukaryotic cells,in particular mammalian cells, as well for the blocking of interactionsbetween mammalian cells, in particular tumor cells, which are mediatedby carbohydrate-binding galectin-3 molecules located on the cell surfaceand are responsible for the development of, in particular, tumordiseases, in particular for the prevention of the formation ofmetastases in mammals.

This technical problem is solved with the methods according to theinvention for the production of pectin hydrolysis products, which resultin the production of oligogalacturonides with a monomer content as lowas possible, a high content of molecules with at least one double bondeach, as well as a degree of esterification of ≧20%, and which can beperformed with a substantially higher yield than with the state of theart.

The problem is solved in particular in that an aqueous solution orsuspension of a pectin or pectin-containing, in particular plant,material, preferably a pectin with a high degree of esterification, istreated in a first step with a first pectin-hydrolyzing enzyme A andthen in a second step with a second pectin-hydrolyzing enzyme B. Thisyields a previously defined pectin hydrolysis product that has excellentproperties as a means for reducing or preventing the adhesion for thelife and/or proliferation functions of cells of harmful, for examplepathogenic, allergenic, infectious, or toxic substances or organisms,for example microorganisms, such as yeasts, fungi, germs, bacteria,viruses, spores, viroids, prions.

Enzyme A may be, for example, a pectinlyase (EC 4.2.2.10) orendopolygalacturonase (EC 3.2.1.15), preferably a pectinlyase, however.Enzyme B may be an endopolygalacturonase or a pectinlyase, preferably anendopolygalacturonase, however.

SUMMARY OF THE INVENTION

According to the invention, it was unexpectedly found thatgalacturonides with double bonds in the molecule are especiallyeffective in blocking the adhesion of, for example, pathogenic germs andcell-damaging substances to epithelial cells of the gastrointestinal andgenitourinary tract. In addition, an especially efficient preventionand/or reduction, for example blocking, requires a higher degree ofesterification, in particular ≧20, preferably ≧30, ≧40, ≧50, especiallypreferably ≧60, ≧65, ≧70, or ≧71%. However, the method described in thestate of the art only produces galacturonides that do not have anydouble bonds and are practically completely deesterified.

Within the context of the present invention, the term “degree ofesterification” means that portion of acid groups of a galacturonideprincipally available for esterification that is esterified with analcohol, especially methanol.

Within the context of the present invention, “unsaturated galacturonicacid molecules” are in particular 4,5-unsaturated galacturonic acidmolecules.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In one embodiment of the invention, the treatment with enzyme B isfollowed by a treatment with another, third enzyme C. This enzyme C maybe a pectin esterase (EC 3.1.1.11). This makes it possible to adjust thedegree of esterification of the products in an especially accuratemanner.

Another embodiment of the invention provides that after completedenzymatic treatment according to the invention, the remaining,undissolved components are removed from the solution by centrifugationand/or ultrafiltration.

Another embodiment provides that the solution obtained after completedenzymatic treatment according to the invention and clarification bycentrifugation or ultrafiltration is transformed by one of the actuallyknown methods into its dry form, for example, into a ground, grainy,granulated, or powder form.

The solution obtained after the enzymatic treatment according to theinvention, or the dry product obtained thereof, show a very good effectwith respect to blocking the adhesion of, for example, pathogenic germsand cell-damaging substances to, for example, epithelial cells of thegastrointestinal and genitourinary tract in humans and animals. For thisreason, they may be used in animal feed, for example by pig farmers toprevent diarrhea disorders in piglets.

The solution or suspension of the pectin or pectin-containing,preferably plant, material used according to the invention has a pHvalue in a range from 3.5 to 5.5 or/and in another preferred embodimenta pectin concentration of 3% to 25%.

The treatments with enzymes A, B, and possibly C take place at a pHvalue of 3.5 to 5.5 over a period of 2 hours to 24 hours at atemperature from 25° C. to 60° C. and a concentration of enzyme A, B,possibly C, of 10 to 30 ml/kg of pectin.

Another preferred embodiment provides that the content of galacturonidesin the pectin hydrolysate (% by weight in relation to dry substance) isat least 60, ≧70, ≧75, ≧80, or especially preferably at least 85% byweight.

Another preferred embodiment provides that in the pectin hydrolysate thecontent of carbohydrates with a DP-1 (monomers) in relation to the totalcarbohydrates of the pectin hydrolysate is <25, <20, <10, <8, <5,especially preferably <4% by weight (related to the dry substance).

Another preferred embodiment provides that the content of carbohydrates,in particular galacturonides with a degree of polymerization DP>10 inrelation to the total carbohydrate content of the pectin hydrolysate, isless than 10, <8, especially preferably <5% by weight (related to thedry substance).

Another preferred embodiment provides that the content of unsaturatedgalacturonides in relation to the total content of galacturonides in thepectin hydrolysate is at least 10, preferably >15, >20, >25, >30, inparticular 36.5% by weight to 46% by weight (related to dry substance).

In a preferred embodiment, the pectin hydrolysates produced according tothe invention have a content of at least 60, ≧70, ≧75, ≧80, or,especially preferably, at least 85% (related to dry substance) ofcarbohydrates, in particular galacturonides with a degree ofpolymerization of 2 to 10, preferably 3 to 8, especially preferably 4.5(% by weight dry substance, related to the total carbohydrate content ofthe pectin hydrolysate).

In an especially preferred embodiment, the pectin used is citrus pectin,apple pectin, or sugar beet pectin.

In a preferred embodiment, the pectin-containing material used, inparticular pectin-containing plant material, is apple pomace, sugar beetcossettes, or citrus pellets, i.e., dried residues, for example, fromthe production of orange juice, lemon juice and/or lime juice.

The pectin hydrolysates produced according to the invention, i.e.,pectin hydrolysis products, are excellently suited as a pharmaceuticalor dietetic preparation for fighting infectious diseases or/and forfighting the adhesion of harmful substances and/or organisms tomammalian cells, in particular human cells.

The pectin hydrolysates produced according to the invention are alsoexcellently suited as a pharmaceutical preparation for inhibitingcell-cell interactions and/or interactions between cells and theextracellular matrix in humans or mammals, in particular suchinteractions mediated by galectin-3 molecules located on the cellsurface. The pectin hydrolysis products according to the invention aretherefore particularly suited for inhibiting cell-cell and/orcell/matrix interactions involving tumor cells, and which are thereforeresponsible for the development of diseases, especially tumor diseases,in humans or mammals. The pectin hydrolysates according to the inventiontherefore are also suitable as a pharmaceutical preparation for treatingtumor diseases, especially for reducing the formation of metastases incancers, since they prevent the galectin-3-mediated tumor cell adhesionand/or the invasive potential of the tumor cells.

The invention therefore also relates to the pectin hydrolysates obtainedaccording to the invention, i.e., pharmaceutical preparations anddietetic preparations containing pectin hydrolysis products, which maybe, for example, foods or snacks, such as dairy products, yogurt,cereals, baked goods, etc.

The invention also relates to the use of the previously mentioned pectinhydrolysis products for the production of pharmaceuticals that preventthe attachment or adhesion of harmful substances and/or organisms tomammalian cells, in particular human cells, in particular for fighting,i.e., prophylaxis and therapy, of infectious diseases, poisonings,allergies, etc.

The invention furthermore relates to the use of the previously mentionedpectin hydrolysis products for preventing the attachment or adhesion ofharmful substances and/or organisms to mammalian cells, in particularhuman cells, in particular for fighting infectious diseases, poisonings,allergies, etc.

The infections fought according to the invention may be infections ofthe blood system, respiratory tract, genitourinary tract, nasopharyngealspace, or gastrointestinal tract.

Another field of use is human nutrition, where they are helpful, forexample, in preventing diarrhea in infants, but also in adults.

The invention also relates to the use of the previously mentioned pectinhydrolysis products for inhibiting cell-cell interactions and/orinteractions between cells and the extracellular matrix, in particularof such interactions mediated by carbohydrate-binding galectin-3molecules located on the cell surface and which are responsible for thedevelopment of human and mammalian diseases, especially tumor diseases.These diseases include, in particular, prostate cancers, kidney cancers,Kaposi sarcomas, forms of chronic leukemia, breast cancers, breastadenocarcinomas, sarcomas, ovarian cancers, rectal cancers, throatcancers, melanomas, tumors of the small intestines, colon cancers,bladder tumors, mastocytomas, lung cancers, bronchial cancers,pharyngeal squamous cell carcinomas, gastrointestinal cancers, andstomach cancers. The pectin hydrolysis products according to theinvention may be used in particular to reduce the invasive potential ofmetastasizing tumor cells and/or to inhibit the adhesion of tumor cells.It is preferred that the pectin hydrolysis products produced accordingto the invention are administered orally.

Another preferred embodiment of the invention relates to the use of thepectin hydrolysis products according to the invention for treating tumordiseases in humans or mammals. The pectin hydrolysates according to theinvention preferably may be used to treat tumors based on cell-cellinteractions and/or interactions between cells and the extracellularmatrix, in particular interactions mediated by carbohydrate-bindinggalectin-3 molecules located on the cell surface. By using the pectinhydrolysates according to the invention, preferably prostate cancers,kidney cancers, Kaposi sarcomas, forms of chronic leukemia, breastcancers, breast adenocarcinomas, sarcomas, ovarian cancers, rectalcancers, throat cancers, melanomas, tumors of the small intestines,colon cancers, bladder tumors, mastocytomas, lung cancers, bronchialcancers, pharyngeal squamous cell carcinomas, gastrointestinal cancers,and stomach cancers can be treated. The use of the pectin hydrolysatesaccording to the invention for treating tumors specifically aims for theinhibition of adhesion of tumor cells and/or reduction of the invasivepotential of metastasizing tumor cells.

The invention also relates to the use of the previously mentioned pectinhydrolysis products for producing a pharmaceutical preparation forinhibiting cell-cell interactions and/or interactions between cells andthe extracellular matrix, in particular interactions that are mediatedby carbohydrate-binding galectin-3 molecules located on the cell surfaceand that are responsible for the development of human or mammaliandiseases, in particular of the previously described tumor diseases. Thepharmaceutical preparation for inhibiting cell-cell interactions and/orinteractions between cells and the extracellular matrix are preferablyadministered orally.

Another preferred embodiment of the invention therefore relates to theuse of the pectin hydrolysis products according to the invention forproducing a pharmaceutical preparation that may be used to treat thepreviously described tumor diseases, i.e., for tumors based on cell-cellinteractions and/or interactions between cells and the extracellularmatrix, especially interactions that are mediated bycarbohydrate-binding galectin-3 molecules located on the cell surface.According to the invention it is provided that the pharmaceuticalpreparation may be used for reducing tumor growth and/or for reducingthe formation of metastases, whereby the pharmaceutical preparationaccording to the invention prevents in particular the adhesion of tumorcells and/or reduces the invasive potential of tumor cells. Thepharmaceutical preparation according to the invention is preferablyadministered orally.

The present invention also relates to a method for blocking the adhesionof harmful, in particular, pathological substances or organisms to cellsof a human or mammalian body, comprising the administration of thepectin hydrolysis products produced according to the invention to ahuman or mammal in a quantity that is sufficient to block the adhesionof the harmful substances or organisms to mammalian cells and to preventthe development of an infection. The pectin hydrolysis products producedaccording to the invention are preferably administered orally.

The invention also relates to a method for inhibiting cell-cellinteractions and/or interactions between cells and the extracellularmatrix that are mediated by carbohydrate-binding galectin-3 moleculeslocated on the cell surface and that are responsible for the developmentof human or mammalian diseases, in particular the previously mentionedtumor diseases, comprising the administration of the pectin hydrolysisproducts produced according to the invention to a human or mammal with atumor disease in a quantity that is sufficient to reduce and/or toinhibit galectin-3-mediated cell-cell interactions and/or cell-matrixinteractions. The pectin hydrolysis products produced according to theinvention are preferably administered orally.

The present invention also relates to pharmaceutical preparations thatcontain the pectin hydrolysis products according to the invention inpharmaceutically or therapeutically effective quantities. In the contextof the present invention, a “pharmaceutical preparation” is a mixtureused for diagnostic, therapeutic, and/or prophylactic purposes thatcontains the pectin hydrolysis products according to the invention asactive ingredients in a form that can be well administered in patientsor mammals. The pharmaceutical preparation may be a solid or a liquidmixture. The expression “in pharmaceutically or therapeuticallyeffective quantities” means that the active ingredients are contained inthe pharmaceutical preparation in a dose that is sufficient to preventthe outbreak of a disease, for example, of an infectious disease ortumor disease, to heal the condition of such a disease, to stop theprogression of such a disease, and/or to relieve the symptoms of such adisease. In addition to the pectin hydrolysis products according to theinvention, the pharmaceutical preparations according to the invention ina preferred embodiment also contain pharmaceutically compatiblevehicles, as well as diluents, release agents, lubricants, adjuvants,fillers, sweeteners, aromas, coloring agents, flavoring agents, or otherpharmaceutically effective substances.

The dietetic preparations according to the invention also contain thepectin hydrolysis products in a pharmaceutically effective quantity.

Other preferred embodiments are described in the secondary claims.

The invention is explained in more detail in the following examples.

Example 1

0.3 ml of a pectinlyase (for example, Rohapect PTE by Röhm) were addedto 1 l of citrus pectin solution (30 g highly esterified pectin in 1 lof water), and the solution was incubated with stirring at a pH 5.0 and45° C. for 2 hours. Then 0.75 ml of an endopolygalacturonase (forexample, Pectinase PL by Amano) were added and incubated under the samereaction conditions for another 3 hours. Then the enzymes weredeactivated by heating to 95° C. The insoluble residue was removed bycentrifugation, the clear solution was evaporated until dry, and theobtained solid was weighed. The weight was 25.8 g (corresponding to ayield of 75.6% related to used raw material).

The resulting product was analyzed using generally known analysismethods, and the following composition was determined:

Carbohydrates DP1 3.6% Galacturonides 83.9% of these: unsaturated 46.0%(assumed mean DP = 4.5) DP 2-10 80.4% DP > 10 16.0% Degree ofesterification 72.0% Salt content 3.0% Raw protein 1.7% Water content4.6%

Example 2

0.3 ml of a pectinlyase (for example, Rohapect PTE by Röhm) were addedto 1 l of citrus pectin solution (30 g highly esterified pectin in 1 lof water), and the solution was incubated with stirring at a pH 5.0 and45° C. for 2 hours. Then 0.75 ml of an endopolygalacturonase (forexample, Pectinase PL by Amano) were added and incubated under the samereaction conditions for another 3 hours. Then the enzymes weredeactivated by heating to 95° C.

The insoluble residue was removed by centrifugation and the clearsolution underwent ultrafiltration (cut-off 10,000). The permeate wasdried and yielded 22.8 g of solid matter (a yield of 66.8% related toused raw material).

Carbohydrates DP1 3.0% Galacturonides 84.1% of these: unsaturated 36.5%(assumed mean DP = 4.5) DP 2-10 93.0% DP > 10 4.0% Degree ofesterification 72.0% Salt content 6.7% Raw protein 1.3% Water content4.4%

Example 3

0.3 ml of a pectinlyase (for example, Rohapect PTE by Röhm) were addedto 1 l of citrus pectin solution (30 g highly esterified pectin in 1 lof water), and the solution was incubated with stirring at a pH 5.0 and45° C. for 2 hours. Then 0.75 ml of an endopolygalacturonase (forexample, Pectinase PL by Amano) were added and incubated under the samereaction conditions for another 3 hours. Then 0.5 ml of a pectinesterase (for example, Rheozyme by Novo Nordisk) were added andincubated for another 45 minutes. Then the enzymes were deactivated byheating to 95° C. The insoluble residue was removed by centrifugation,the clear solution was evaporated until dry.

The resulting product was analyzed using generally known analysismethods. In contrast to Example 1, a degree of esterification of 35% wasdetermined.

Example 4

Dried orange peel or citrus pellets were comminuted to a particle sizeof approximately 1-5 mm, and 100 g of this was stirred into 400 ml ofwater and left to soak. Then concentrated nitric acid (10 g) was added,and the suspension was heated to 85° C. and stirred at this temperaturefor 1.5 hours. This was followed by cooling to 45° C.; the pH value wasincreased with NaOH to 4.5, followed, after addition of 0.3 ml of apectinlyase (for example, Rohapect PTE by Röhm), by 2 hours ofincubation. Then 0.75 ml of an endopolygalacturonase (for example,Pectinase PL by Amano) were added and incubated under the same reactionconditions for another 3 hours. Then the enzymes were deactivated byheating to 95° C., concentrated, and the suspension was dried with adrum dryer.

Example 5

Dried orange peel or citrus pellets were comminuted to a particle sizeof approximately 1-5 mm, and 100 g of this was stirred into 400 ml ofwater and left to soak. Then concentrated HCl (8 g) was added, and thesuspension was heated to 85° C. and stirred at this temperature for 1.5hours. This was followed by cooling to 45° C.; the pH value wasincreased with NaOH to 4.5, followed, after addition of 0.3 ml of apectinlyase (for example, Rohapect PTE by Röhm), by 2 hours ofincubation. Then 0.75 ml of an endopolygalacturonase (for example,Pectinase PL by Amano) were added and incubated under the same reactionconditions for another 3 hours. Then the enzymes were deactivated byheating to 95° C., concentrated, and the suspension was dried with adrum dryer.

Example 6 Prevention of Adhesion of Pathogenic Germs in Human EpithelialCells

For this test, human uroepithelian cells obtained by centrifugation frommorning urine as well as two strains of staphylococcus aureus and E.coli were used, each as a suspension with 10⁹ germs/mL.

Test Procedure

Epithelial cells and germ suspension were incubated together at 37° C.for 30 minutes. The epithelial cells then were separated from thenon-adherent germs by membrane filtration (8μ). The filters were washedseveral times, placed into normal saline, and the epithelial cells weresuspended in it. After centrifugation of the suspension in saline, thepellet was applied to slides and stained according to May-Grünwald andGiemsa. The number of germs adhering to 50 epithelial cells werecounted. This number represented the blank value. Epithelial cells towhich no germ solution had been added were used as a control.

In the main test, epithelial first were incubated with aqueous solutionsof various concentrations consisting of pectin hydrolysis productsproduced according to the invention (according to Example 1) for 1, 2 or3 hours. They were then combined with the germ suspension and treatedfurther as described above. The measuring value was obtained by countingthe germs adhering to 50 epithelial cells.

Result

No reduction in the germ adhesion to the epithelial cells was observedfor the “neutral” carbohydrates, such as raffinose, nystose, andisomelezitose used for comparison. By using the pectin hydrolysisproducts according to the invention, the adhesion of all testedmicroorganisms was almost completely prevented (blockage of >95%).

Example 7

1.5 g of the dried permeate from Example 2 were dissolved in 100 ml of50 mM Nucleic acid-acetate solution with a pH value of 5.0 and were thengiven through a column (2.6×30 cm) that had been filled with the anionexchanger AG 1×2 (BioRad) and equilibrated with 50 mM Nucleicacid-acetate solution, pH value 5.0. The forerun from the column wasanalyzed with HPAEC (high performance anion exchange chromatography) andhydrolyzed for one hour with 1 N HCl at 95° C.

Result

In comparison with Raftiline (Orafti) as a standard, theoligosaccharides eluted from the column had a DP distribution of 2-12.

The analysis of the hydrolysates using a sugar analyzer (Biotronik)found primarily galactose (70%) as monosaccharides, as well as arabinose(23%), and traces of glucose and mannose. Overall, 8.3% of thegalacturonide-containing products were obtained as neutralsugar-containing oligosaccharides in the forerun.

Example 8 Growth of Colon Cancer Cell Lines on Extracellular Matrix(ECM) in the Presence of Pectin Hydrolysate

Human colon cancer cell lines HT-29 or Caco-2 were seeded with a celldensity of 1×10⁴ cells/ml in 15 mm Petri dishes and cultivated in mediumRPMI 1640+10% fetal calf serum (FCS) (HT-29) or, respectively, inMEM+10% FCS (Caco-2) at 37° C. under an atmosphere containing 5% CO₂.The cells were left to grow for 1 to 2 days until reaching confluence.The dishes were then washed once with PBS, then incubated with PBS and0.5% Triton X-100 for 30 minutes at room temperature on an agitator, andthen washed 3 times with PBS. The previously described cell lines thenwere again seeded on the dishes with the ECM layers prepared in thisway. The influence of the pectin hydrolysate on cell growth wasdetermined by counting the cells. For this, the cells were again removedafter 48 hours with trypsine/EDTA solution in HBBS (10 min) and werewashed in PBS solution. Then the number of living cells was determinedby staining with trypan blue solution (650 mg trypan blue in 400 ml 0.9%NaCl, 1:1 (v/v)) in a Neubauer counting chamber. As a control,experiments were performed with glucose. The results are shown in Table1.

Table 1 shows that glucose had no influence on the growth of the HT 29and Caco-2 cell lines, while pectin hydrolysate reduced cell growth inrelation to the concentration used by up to 75%.

TABLE 1 Reduction in Cell Growth HT 29 Caco-2 Pectin PectinConcentration hydrolysate Glucose hydrolysate Glucose 0.01% 30% 0% 25%0%  0.1% 45% 1% 43% 0%  1.0% 75% 0% 70% 0%

Example 9 Reduction of Invasive Capacity of Caco-2 Cells with PectinHydrolysate

The effect of pectin hydrolysate on the invasive capacity of Caco-2cells was studied using the invasion test described by Erkell andSchirrmacher (Cancer Research, 48 (1988), 6933-6937). The test is basedon the migration of cells through the pores of a nucleoporepolycarbonate filter in a protein gel that contains several ECMproteins, such as, for example, type 1 and type III collagen,fibronectin, and laminin, to a nitrocellulose filter. The cells wereadded together with the pectin hydrolysate into a test system, afterwhich the cells that had migrated through the protein layer werequantitatively evaluated in the lower nitrocellulose layer. As acontrol, the effect of glucose on the invasive capacity of Caco-2 cellswas studied.

The results of these studies are shown in Table 2. The results show thatthe pectin hydrolysate according to the invention was able to reduce theinvasive capacity of Caco-2 cells in part significantly in relationshipto the concentration used, while glucose only caused a slight reductionof the invasive capacity of Caco-2 cells in higher concentrations.

Reduction Of Invasive Capacity Of Caco-2 Cells Concentration PectinHydrolysate Glucose 0.01% 30% 0%  0.1% 69% 2%  1.0% 88% 3%

Example 10 Anti-Galectin-3 Antibody Binding by Pectin Hydrolysate

The expression of galectin-3 on colon cancer cells was determined withimmunofluorescence/flow cytometry methods using ananti-galectin-3-specific monoclonal antibody (mouse-Ig) and acorresponding anti-mouse FITC-coupled secondary antibody. Increasingconcentrations of the pectin hydrolysate and of glucose as a controlwere incubated together with the primary antibody on the target cells,and then the inhibiting influence of the soluble sugar substance on theanti-galectin-3 binding was measured.

The influence of pectin hydrolysate on the anti-galectin-3 binding isshown in Table 3. Table 3 shows that glucose reduces the bindingreaction of the monoclonal anti-galectin-3 antibody not at all or onlyslightly, while pectin hydrolysate reduces the binding of the antibody,in relation to the concentration used, in part substantially.

TABLE 3 Reduction of Binding Monoclonal Anti-Galectin-3 Antibody HT 29Caco-2 Pectin Pectin Concentration hydrolysate Glucose hydrolysateGlucose 0.01% 34 0 28 0  0.1% 67 0 63 0  1.0% 85 2 82 0

The invention claimed is:
 1. A composition comprising a dairy productcontaining a pectin hydrolysis product having a content ofgalacturonides that include at least one 4,5-unsaturated galacturonicacid molecule, wherein the galacturonic acid molecules areesterified >20% with methanol and wherein the pectin hydrolysis producthas (1) a content of carbohydrates with a DP-1 of <25% by weight basedon the total dry weight of carbohydrates or (2) a content ofgalacturonides of at least 60% by weight on the total dry weight ofcarbohydrates.
 2. The composition according to claim 1, wherein thedairy product is a yogurt.
 3. The composition according to claim 2,wherein the pectin hydrolysis product has a content of galacturonides ofat least 60% by weight based on the total dry weight of carbohydrates.4. The composition according to claim 2, wherein the pectin hydrolysisproduct has a content of galacturonides that include at least 10% inrelation to the total dry weight of galacturonides of 4,5-unsaturatedgalacturonic acid molecules.
 5. The composition according to claim 2,wherein the galacturonides are esterified ≧30% with methanol.
 6. Thecomposition according to claim 2, wherein the pectin hydrolysis producthas a content of galacturonides with a degree of polymerization DP>10 inrelation to the total carbohydrate content of the pectin hydrolysate isless than 10% by weight related on the dry substance.
 7. The compositionaccording to claim 2, wherein the pectin hydrolysis is produced bytreating a pectin or pectin-containing plant material in aqueoussolution or suspension with a pectinlyase and then with anendopolygalacturonase, and after said enzyme treatments, recovering thepectin hydrolysis product.
 8. The composition according to claim 1,wherein the pectin hydrolysis product has a content of carbohydrateswith a DP-1 of <25% by weight based on the total dry weight ofcarbohydrates.
 9. The composition according to claim 4, wherein thepectin hydrolysis product has a content of galacturonides of at least60% by weight based on the total dry weight of carbohydrates.
 10. Thecomposition according to claim 1, wherein the pectin hydrolysis producthas a content of galacturonides of at least 60% by weight based on thetotal dry weight of carbohydrates.
 11. The composition according toclaim 1, wherein the pectin hydrolysis product has a content ofgalacturonides that include at least 10% in relation to the total dryweight of galacturonides of 4,5-unsaturated galacturonic acid molecules.12. The composition according to claim 1, wherein the galacturonides areesterified ≧30% with methanol.
 13. The composition according to claim 1,wherein the pectin hydrolysis product has a content of galacturonideswith a degree of polymerization DP>10 in relation to the totalcarbohydrate content of the pectin hydrolysate is less than 10% byweight related on the dry substance.
 14. The composition according toclaim 1, wherein the pectin hydrolysis is produced by treating a pectinor pectin-containing plant material in aqueous solution or suspensionwith a pectinlyase and then with an endopolygalacturonase, and aftersaid enzyme treatments, recovering the pectin hydrolysis product.
 15. Amethod which comprises combining a pectin hydrolysis product having acontent of galacturonides that include at least one 4,5-unsaturatedgalacturonic acid molecule, wherein the galacturonides are esterified≧20% with methanol, with a dairy product.
 16. The method according toclaim 15 in which the pectin hydrolysis product has a content ofgalacturonides that include at least 10% in relation to the total dryweight of galacturonides of 4,5-unsaturated galacturonic acid molecules,and wherein the pectin hydrolysis product has (1) a content ofcarbohydrates with a DP-1 of <25% by weight based on the total dryweight of carbohydrates or (2) a content of galacturonides of at least60% by weight based on the total dry weight of carbohydrates or both (1)and (2).
 17. The method of claim 16 in which the dairy product isyogurt.
 18. The method of claim 15 in which the dairy product is yogurt.